The blood coagulation cascade or system is comprised of a group of zymogens that are converted by limited proteolysis to active enzymes. This active cascade of enzymes ultimately forms a fibrin clot from fibrinogen. This blood clotting cascade is divided into two pathways, extrinsic and intrinsic. The series of reactions that convert the zymogens to enzymes requires a variety of protein cofactors such as Blood Factors VIII and V. In turn these cofactors are regulated by a number of other proteins such as Protein S and Protein C. High, K.A., Antithrombin III, Protein C, and Protein S, Arch. Pathol. Lab. Med. (1988); Vol. 112:pp.28-36.
Protein S is a naturally occurring anticoagulant protein. It circulates in two forms--free and bound to C4B. Approximately 40% of the protein is found in the free form while 60% exists in the bound form. Only the free form has functional activity. Blanc, P., et al., Deficit Constitution en Proteine S a l'Origine de Thrombose Vasculaire Digestive, La Presse Medical (1990): Vol. 19: pp.416-419. Protein C is also an anticoagulant.
Protein S and Protein C exert their effect on the intrinsic pathway of the clotting cascade system. Protein S does not require activation by another factor, however, it is active only in the presence of activated Protein C which is activated by thrombin (Factor IIa). Activated Protein C acts as an anticoagulant by inactivating Factor V and VIII. Protein S increases the anticoagulatory effect of Protein C.
Von Willebrand Factor decreases the inactivation of Factor VIII by activated Protein C and Protein S and that effect is enhanced by the active site of Factor IXa. The von Willebrand Factor acts by binding Factor VIII, thereby protecting it from inactivation. Rick, M.E., Factor IXa and von Willebrand Factor Modify the Inactivation of Factor VIII by Activated Protein C, Journal Lab. Clin. Med. (1990); Vol. 115(4): pp. 415-421.
Deficiency of Protein S has been associated with a number of disease states. For example, individuals who have reduced Protein S levels have an increased risk of venous thromboembolism. In fact, Protein S deficiency is responsible for 8-10% of the cases of venous thromboembolism occurring in young people. Preda, L. et al., A Prothrombin Time-Based Functional Assay of Protein S, Thrombosis Research (1990); Vol. 60:pp.19-32.
There are two types of Protein S deficiency. The first type is associated with mildly reduced levels of total Protein S, but markedly reduced levels of free Protein S, while the second type of Protein S deficiency has markedly reduced levels of both free and total Protein S Woodhams, B.J., et al., Functional Protein S Assay Shows Improved Correlation with Clinical Symptoms in Hereditary Deficiency, Thrombosis Research (1990); Vol. 57:pp.651-657. The only known treatment for Protein S deficiency is lifelong therapy with sodium warfarin. High, K.A., Antithrombin III, Protein C, and Protein S, Arch. Pathol. Lab. Med. 1988; 112:pp.28-36.
There are a number of methods available to measure Protein S levels in individuals. These methods can be divided into two classes. The first class, Protein S antigen level assays, measures both free and bound (total) Protein S. The second class, functional Protein S assays, measures only free Protein S since only free Protein S has any functional activity.
In the antigen level assays, Protein S levels (total Protein S) are measured using a variety of techniques including: ELISA, RIA, IRMA, and electroimmunodiffusion (Laurell Rocket Technique). All of these techniques use polyclonal antibodies to the antigen. One difficulty associated with these methods, however, is that the relative affinities of the antibodies to the free and bound Protein S must be known in order to determine the actual concentration of functional Protein S. Therefore, antigen level assays are of limited value since they cannot unequivocally distinguish the functionally active or free form of Protein S from the bound form which is not functionally active. Bertina, R., Specificity of Protein C and Protein S Assays, Res. Clin. Lab. (1990); Vol. 20:pp127 at 132-134. Moreover, this measurement of the total Protein S antigen does not distinguish between the the two types of Protein S deficiencies. Furthermore, antigen level assays require high sample dilutions and long incubation times.
Alternatively in the functional Protein S assays, functional or free Protein S activity levels are measured using clotting times or thromboplastin times. One functional Protein S assay is based on a prolongation of Factor Xa initiated clotting times. In this assay, Protein S which requires activated Protein C, acts as a co-factor for the activated Protein C to inactivate Factor Va which therefore prolongs clotting times. Some of the other functional Protein S assays activate the Protein C using a snake venom activator. Another functional Protein S assay is based on the activated partial thromboplastin time (APTT). This method is based on the need for functional Protein S to act as a co-factor for the activated Protein C dependent inhibition of blood coagulation. A primary problem associated with this type of test is its inapplicability to patients who are receiving anti-coagulants such as heparin. Since most patients requiring Protein S tests are using oral anticoagulants at the time of the sample collection, these types of tests cannot be used accurately. Bertina, R., Specificity of Protein C and Protein S Assays, Res. Clin. Lab.; 20:p.127. Another problem associated with the APTT type assay is that it relies upon the presumption that normal plasma will give a 100% correction in thromboplastin time. It has been recognized that there is a dependence of prolongation of clotting times on activated Protein C concentration. Thus, persons performing the assay must construct a full standard curve each time the assay is run. Moreover, the assay is very costly and a long time is needed for measurement, while the coagulation time may only be extended by 10 seconds. These factors leave room for error and reduce lab efficiency.
To solve many of the above problems, we have created a test using chromogenic substrates to measure activity levels of functional Protein S. Chromogenic substrates have previously been used in the determination of Factor VIII levels. These Factor VIII assays recognize that Factor Xa concentrations can be linked to Factor VIIIa concentrations by exploiting the coagulation cascade system (Dade.RTM. Factor VIII Chromogenic Assay: Baxter Diagnostics Inc.). Protein C concentrations also have been determined with chromogenic substrates which are cleaved by activated Protein C making a direct and simple measurement for Protein C. High, K.A., Antithrombin III, Protein C, and Protein S, Arch. Pathol. Lab. Med. (1988); Vol. 112:pp.28-36. Since Protein S is not an enzyme, however, it cannot be measured directly using chromogenic substrates. Thus, this invention exploits the relationship of Protein S with other blood coagulation proteins.
The method of the present invention provides an assay which quantitatively determines only the functional levels of Protein S and does not utilize expensive antibody technology to determine the Protein S concentration. This method has an advantage over other Protein S assays in that this assay does not have the problems associated with arbitrarily designating normal pools as having a clotting of 100% and basing other samples on that normal pool value. This is especially true since pregnancy lowers the free Protein S activity. Suzuki, K. et al., Plasma Protein S Activity Measured Using Protac, A Snake Venom Derived Activator of Protein C, Thrombosis Research (1988); Vol. 49:pp.241-251. including a sample from a pregnant woman into the pool would obviously lower the final pool value and distort results.
This invention provides a highly sensitive, reproducible, and convenient assay for determination of the levels of Protein S contained in blood serum, plasma, and other fluids. The invention recognizes that the effect of von Willebrands factors must be eliminated to obtain accurate and reproducible results. Again, since Protein S is not an enzyme, its functional concentration cannot be measured directly by way of a chromogenic substrate. Instead, the ability of Protein S to enhance the inactivation of Factor VIII by activated Protein C is exploited. Residual Factor VIII is activated and then acts wi&:h Factor IXa to activate Factor X. An indicator such as a chromogenic substrate to Factor Xa is then converted by Factor Xa to a measurable signal molecule. The signal molecule can then be related to the amount of Protein S in the sample.